Device and method for the detection of microorganisms which produce low-molecular-weight metabolites

ABSTRACT

Selective detection of microorganisms is achieved by a screening technique in which radioactively labeled, low-molecular-weight metabolites produced by microorganisms to be detected reach an adsorption medium through a semipermeable medium which passes the labeled metabolites but blocks the labeled incubation medium. The adsorption medium is then subject to analysis, such as autoradiography, to detect the presence of the labeled metabolites. An analytical assembly for such detection includes a filter membrane support for growing microorganisms in discrete colonies and for holding a radioactively labeled incubating medium applied thereon, a semipermeable filter medium covering the support having the grown microorganisms and the incubating medium for passing low-molecular-weight, labeled metabolites produced by microorganisms of interest while blocking the incubating medium, and a cohesive layer of an adsorption medium conformably disposed with the filter membrane support and the semipermeable medium and being capable of adsorbing the labeled metabolites which have traversed the semipermeable medium during incubation of the assembly. Following incubation of the assembly, the adsorption medium layer is transferable for analysis, e.g., by autoradiography, to detect the presence of labeled metabolites produced by microorganisms of interest and to identify specific colonies of the microorganisms producing the radioactively labeled metabolite.

This application is a division of application Ser. No. 07/781,023, filedon Oct. 18, 1991.

BACKGROUND OF THE INVENTION

This invention relates to analytical devices and methods for thedetection of microorganisms which produce low-molecular-weightmetabolites.

As disclosed by J. L. Firmin et al., "The Biochemical Pathway for theBreakdown of Methyl Cyanide (Acetonitrile) in Bacteria" BiochemicalJournal., Vol 158 (1976), pp. 223-229, it is known that metabolitesformed in a microbiological or biochemical process can be detected asfollows: a radioactively labeled substance is admixed to a cellsuspension; the mixture is incubated for a certain time; the cells areremoved; and a radioactively labeled metabolite is detected, e.g., byliquid-phase scintillation measurement. This widely used detectionmethod is complicated and time consuming. Moreover, as radioactivelylabeled metabolites are detected independent of their molecular weight,this method is unsuited for the selective detection of microorganismswith specific metabolic performance.

Disclosed in published European patent application, EP-A 124 285, is adetection method for microorganisms in medical test samples. Thismethod, too, suffers from the drawback of registering the totality ofradioactively labeled metabolites, making it unsuited as a method forscreening metabolites produced by specific microorganisms.

SUMMARY OF THE INVENTION

The invention provides for detection of microorganisms on the basis oftheir ability to produce a low-molecular-weight metabolite. Inaccordance with the invention, microorganisms grown on a support, suchas a filter membrane, are provided with an incubating medium containinga radioactively labeled substance which can be metabolized into alow-molecular-weight metabolite by the microorganisms. A semipermeablemedium is provided to cover the support having the grown microorganismsand the incubating medium. An adsorption medium is positioned adjacentthe semipermeable medium so as to be exposed to material passing throughthe semipermeable medium. The semipermeable medium is selected to bepermeable to the low-molecular-weight metabolite but not to the labeledsubstance in the incubation medium. The adsorption medium is selected tobe able to adsorb the low-molecular-weight metabolite. The assemblycomprising the support having the microorganisms and the incubationmedium, the semipermeable medium and the adsorption medium is incubatedfor a time sufficient to allow production of a detectable amount oflow-molecular-weight metabolite if microorganisms of interest arepresent, and the adsorption medium is analyzed, e.g., byautoradiography, for the presence of a labeled metabolite. Preferably,the adsorption medium is in the form of a cohesive layer that cantransferred to an autoradiography film to allow identification ofspecific colonies of microorganisms producing the labeled metabolite.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic, exploded cross section of a device assembly inaccordance with a preferred embodiment of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In accordance with the invention, microorganisms are selectivelydetected via the detection of low-molecular-weight metabolites formed ina microbiological process, a radioactively labeled substance beingmetabolized by microorganisms to produce a radioactively labeledmetabolite, and such metabolite being detected by a determination ofradioactivity. This technique is advantageously applied to the detectionof metabolites whose molecular weight does not exceed approximately 500,and is particularly effective for the detection of metabolites whosemolecular weight does not exceed approximately 250.

Among suitable radioactive labeling materials are the isotopes ¹⁴ C, ³H, ³⁵ S, and ³² P. Other biologically suitable radioactive isotopes maybe used. Preferred among the above-mentioned isotopes are ³ H and ¹⁴ C.Typically, the specific radioactivity of a radioactively labeledsubstance is approximately in the range of 1 millicurie/millimole to 20curie/millimole.

Conveniently, when a substance which is radioactively labeled with ³ Hor ¹⁴ C is metabolized by microorganisms, a low-molecular-weightmetabolite is formed which is respectively labeled with either ³ H or ¹⁴C. Thus, for ³ H or products labeled with ¹⁴ C such as carbon dioxide,ethanol, lactic acid, acetic acid, formic acid, acetone, butanol,butanediol, acetoine or other metabolites. Of particular importance aresubstances labeled with ³ H which are metabolized into ³ H₂ O andsubstances labeled with ¹⁴ C which are metabolized into ¹⁴ CO₂.Radioactively labeled metabolites can be detected by conventionalmethods, e.g., autoradiography or liquid-phase scintillationmeasurement. The use of autoradiography is preferred.

An exemplary detection method in accordance with the invention may beillustrated with reference to FIG. 1. Shown in FIG. 1 are the principalcomponents of an assembly comprising a support 1 (e.g., a known filtermembrane such as a cellulose filter) on which colonies 2 ofmicroorganisms are grown, a semipermeable filter 3 permeable tometabolites of the microorganisms which are of interest, and anadsorption medium 4.

Conveniently, for the growth of colonies 2, the support 1 is placed on astandard agar medium, e.g., platecount agar, available from DifcoLaboratories, USA. When the microorganism colonies 2 have reached adiameter of approximately 0.1 to 5 millimeters, and preferably 0.5 to 2millimeters, these colonies are covered with an incubating solutioncomprising a radioactively labeled substance which can be metabolized bymicroorganisms of interest to produce a labeled, low-molecular-weightmetabolite. To minimize convection in the incubating solution, aviscosity-increasing material may be added, e.g., agar.

Preferably, a hydrophilic sterile filter is used as the support 1,having a pore size of approximately 0.01 to 2 micrometers. For example,such sterile filters may be formed with cellulose nitrate, celluloseacetate, regenerated cellulose, nylon, or some other standardhydrophilic sterile filter material having an appropriate pore size.

The incubating solution may be applied while the support 1 remains on asolid nutrient substrate. Alternatively, it may be advantageous toseparate and transfer the support 1 with the microorganism colonies 2away from the substrate, especially to prevent undue dilution in thedetection of ³ H₂ O.

After the microorganisms 2 have been covered with the incubatingsolution, a semipermeable filter 3 is provided over the support coveringthe microorganisms 2 and the incubating solution (not shown). Thesemipermeable filter 3 allows passage of the low-molecular-weightmetabolite but not the labeled substance contained in the incubatingsolution. Preferably, the semipermeable filter 3 is (or has beenrendered) hydrophobic. For selective permeability to metabolites, thepore size of the semipermeable filter 3 is advantageously in the rangeof approximately 0.02 to 2 micrometers and preferably in the range ofapproximately 0.02 to 0.2 micrometer. Preferably, the semipermeablefilter 3 is selected from the following: Goretex® filters(polytetrafluoroethylene with 0.2- to 0.02-micrometer pore size,disposed on a polyester supporting fabric), glass-fiber filters havingappropriate pore sizes and rendered hydrophobic (e.g., by silanetreatment), and silicone membranes having appropriate pore sizes and athickness in the range of approximately 0.1 to 1 millimeter.Alternatively, a semipermeable layer may be made by depositing ahydrophobic powder material, e.g., Aerosil R972 available from Degussa,Germany.

To prevent mixing of the microorganisms of the colonies 2, a supportingfabric 5 may be placed between the support 1 and the semipermeablefilter 3. Such a supporting fabric may consist of a net-like cottonfabric, such as gauze. Instead of the support fabric or in additionthereto, another filter membrane, similar to the filter membrane ofsupport 1, for example, may be placed between support 1 andsemipermeable filter 3.

An adsorption medium 4 is placed on the semipermeable filter 3 for theadsorption of the low-molecular-weight metabolite passing through thesemipermeable filter 3. In the case where ³ H₂ O is being detected, aglass substrate covered with a molecular sieve or with silica gel isadvantageously used as adsorption medium. For the detection of ¹⁴ CO₂, aglass substrate covered with lime is preferred. Specifically, amolecular sieve having a pore size in the range of approximately 3 to 4Angstroms, or, alternatively a silica gel, may be deposited on a glasssubstrate to a thickness in the range of approximately 0.1 to 5millimeters. As will be known to those skilled in the art, otheradsorbing materials may be used as the adsorption medium, such as gypsumor aluminum oxide.

For added stability of the adsorption medium 4 it may be desirable touse a binder material, e.g., derivatized cellulose, starch, cement,water glass, glass fibers, or any other suitable binder material as iscustomarily used in the preparation of thin-film chromatographysubstrates.

The resulting assembly comprising the support 1 with the microorganismcolonies 2 and the applied incubating solution (not shown), thesupporting fabric 5 (if any), the semipermeable filter 3, and theadsorption medium 4 is incubated for approximately 1 to 12 hours at anappropriate temperature in the range of approximately 5 to 110 degreesC. to allow formation of a detectable amount oflabeled,low-molecular-weight metabolite if microorganisms of interestare present. Known incubating media with appropriate radioactivelabeling materials may be used. Examples of such incubating media aredescribed hereinbelow. By application of a weight or other compressingmeans, the thickness of the assembly may be reduced to approximately 5millimeters, for example. The length of the assembly may beapproximately 5 centimeters.

Because the diffusion rate of low-molecular-weight metabolites varies indifferent semipermeable media, the preferred incubation time depends onthe type of semipermeable filter which is being used. When a siliconemembrane is used as the semipermeable filter 3, the preferred incubationtime is up to approximately 3 hours. In the case where Goretex® or ahydrophilic glass-fiber filter is used as the semipermeable filter 3,the preferred incubation time is up to approximately 1 hour. Thepreferred incubation temperature depends on the temperaturecharacteristics of the microorganisms being detected. For example, forthe detection of mesophile microorganisms, the preferred incubationtemperature is in the range of approximately 30 to 37 degrees C.

After incubation the adsorption medium is removed and may be treatedwith a suitable scintillator material, such as Enhance®, available fromDuPont, USA. Other suitable scintillator solutions are described by B.R. Bochner et al., Anal. Biochem., Vol. 131 (1984), pp. 510-515.

The invention provides for highly sensitive detection of microorganismswhich metabolize a radioactively labeled substance into a metabolitewhich can be selectively detected in the manner described above. Incontrast with known techniques, the microorganism detection technique ofthe invention is also applicable to colonies of microorganisms grown inlarge numbers on the surface of a solid nutrient medium.

The technique in accordance with the invention is advantageously usedfor the detection of microorganisms which produce low-molecular-weightmetabolites, and especially for the detection of microorganisms whichare capable of converting bound methyl groups into a correspondingalcohol, aldehyde or carbonic acid. For example, the method can be usedfor the detection of microorganisms capable of oxidizing the labeledmethyl groups of 5-methyl-2-chloropyridine into a corresponding acid andlabeled water.

In the following examples, the physical quantities described therein areunderstood to be nominal or approximate.

Example 1. Alcaligenes eutrophus (DSM 428) and Pseudomonas aeruginosa(DSM 50071) were cultivated separately in a nutrient broth, availablefrom Oxoid, USA, at 30 degrees C., overnight on a shaking machine. Then,one part Pseudomonas culture was mixed with 999 parts Alcaligenesculture, and 0.1 milliliter of this mixture was spread in 10⁻⁴ -dilutiononto a cellulose nitrate filter, available from Sartorius, USA. Thecellulose nitrate filter was placed on plate count agar (PCA), availablefrom Difco, USA. The filter had a diameter of 5 centimeters and a poresize of 0.2 micrometer. Overnight, the cells grew into approximately1000 colonies with diameters from 0.5 to 1 millimeter. The cellulosenitrate filter supporting the colonies was lifted from the PCA-mediumand placed in a Petri dish with the colonies facing up. The filtersupport with the colonies was soaked with 0.18 milliliter radioactivesolution having the following composition: 5 gram/liter melted agar; 5gram/liter Tryptone, available from Difco, USA; 2.5 gram/liter yeastextract, available from Difco, USA; 2 millimose D-[6-³ H(N)]-glucose, at0.1 curie/millimose, available from NEN/DuPont, USA. Immediatelythereafter, a second cellulose filter support, which had been moistenedby contact with plate count agar was placed on the colonies, and ahydrophobic TE35-filter (polytetrafluoroethylene on polystyreneavailable from Schleicher & Schuell, Germany) with a diameter of 8centimeters and a pore size of 0.2 micrometers was placed on the secondcellulose nitrate filter support. Thereafter, a water-specificadsorption plate was immediately deposited with the adsorption layerfacing down so as to be exposed to material passing through theTE35-filter. The adsorption plate had been produced as follows: 8 gramsof 4-Angstrom molecular sieve powder with grain size from 2 to 3micrometers was suspended in 7 milliliters of water, and 0.1 gram ofglass fiber was then added. The glass fiber consisted of finely groundGF/D-filter (glass-fiber filter, Type D, available from Whatman, USA).There followed degassing of the assembly in a vacuum. The pulpy mass waspoured onto a roughened 6-by-6-centimeter glass substrate, dried for 1hour at 70 degrees C., and activated for 4 hours at 150 degrees C. in ahigh vacuum. The resulting layer had a thickness of approximately 2millimeters. The substrates were stored over phosphorus pentoxide.

The resulting assembly was squeezed down to a thickness of approximately5 millimeters by application of a 450-gram stamper. The compressedassembly was then incubated for 1 hour at 30 degrees C. Subsequently,the molecular sieve adsorption plate was removed and the adsorptionlayer thereon immediately sprayed with Enhance®. Autoradiography wasthen carried out according to the directions given by R. A. Laskey,Radioisotope Detection by Fluorography and Intensifying Screens,Amersham, United Kingdom, Review 23, 1984. The duration of the exposurewas 2 days. Scattered Pseudomonas colonies showed up as black spots witha diameter of 5 millimeters. The dominant background of the Alcaligenescolonies remained invisible. Pseudomonas metabolizes tritium-treatedglucose to produce ³ H₂ O but Alcaligenes does not metabolize glucose.

Example 2. The procedure was the same as in Example 1 above, except forthe following modifications: Instead of using the ³ H-glucose, D-[¹⁴C(U)]-glucose, also available from NEN/DuPont, USA, at 3 curie/mole wasused as the radioactive incubating medium. A hydrophobic filter was madeby baking a glass-fiber filter, CF/A available from Whatman, USA, withRepel-Silan, available from LKB, Sweden, at 160 degrees C. until dry.The resulting pore size and layer thickness of the hydrophobic filterwere 1.4 micrometer and 0.27 millimeter, respectively.

A CO₂ -specific adsorption medium was made as follows: lime tablets,available from Drager, Switzerland, were finely ground by mortar andpestle and formed into a paste with water. The paste was spread out witha spatula to form a 2-millimeter layer on a 6-by-6-centimeter glasssubstrate, and subsequently dried in an desiccator over NaOH tablets forseveral days. Further processing was as in Example 1.

The glucose-metabolizing Pseudomonas colonies produced ¹⁴ CO₂ and showedup as 5-millimeter black spots. The Alcaligenes colonies remainedinvisible.

Example 3. The procedure was the same as in Example 1 above, with thefollowing modifications: Instead of plate count agar, a carbon-freemineral medium was used, as described by H. Kulla et al., Arch.Microbiol., Vol. 135 (1983), pp. 1-7. A filter membrane of regeneratedcellulose, available from Sartorius, Germany, was inoculated directly byspreading of biomass from the aerobic stage of a waste treatment plant.Gaseous xylol formed in an desiccator was used as a source of carbon andof energy. Colonies having a diameter of 1 to 2 millimeters are formedwithin a week. The radioactive incubating solution included 0.5% moltenagar and 5 millimole 2-Cl-5-(methyl-³ H)-pyridine at 10 curie/millimole.The hydrophobic filter consisted of a 0.17-millimeter-thick siliconemembrane made from Sylgard 18, available from Dow Corning, USA. Asupporting fabric consisting of a layer of commercially availablemedical gauze was placed between the colonies and the silicone membrane.The incubation period was 3 hours. The adsorption medium consisted of aPSC-finished substrate coated with silica gel 60 to a thickness of 2millimeters without fluorescence indicator, available from Merck,Germany. Otherwise, processing was as in Example 1.

Colonies capable of oxidizing the methyl group in the2-chloro-5-methyl-pyridine showed up as dark spots, removable from thecellulose support filter by customary microbiological methods.

While the foregoing illustrative examples used discrete microbialcolonies grown on a support, which provided the advantage of allowingidentification and quantification of the colonies having the metaboliccapability being studied, it will be recognized that discrete coloniesare not required simply to learn whether microorganisms capable ofmetabolizing the labeled substances are present in a sample.

I claim:
 1. A device for the detection of microorganisms which produce alow-molecular-weight metabolite comprising:a semipermeable medium havinga pore size in the range from 0.01 to 2 micrometers which is permeableto the low-molecular weight metabolite; a support for growingmicroorganisms thereon and for holding a radioactively labeled substancefrom which the metabolite can be produced by the microorganisms beingdetected, the support being positioned at a first side of thesemipermeable medium; and an adsorption medium positioned at a secondside of the semipermeable medium opposite to the first side, theadsorption medium being able to effectively adsorb the metabolite andbeing transferable for analysis to detect the presence of radioactivelylabeled metabolite.
 2. The device of claim 1, wherein the supportcomprises a hydrophilic sterile filter membrane.
 3. The device of claim1, wherein the semipermeable medium comprises hydrophobic materialhaving a pore size in the range from 0.02 to 2 micrometers.
 4. Thedevice of claim 3, wherein the pore size the hydrophobic material is inthe range from 0.02 to 0.2 micrometer.
 5. The device of claim 1, whereinthe adsorption medium comprises a material selected from the groupconsisting of a molecular sieve, silica gel, lime, gypsum, and aluminumoxide.
 6. The device of claim 1, further comprising a supporting mediuminterposed between the support and the semipermeable medium.
 7. Thedevice of claim 1, wherein the support comprises a filter membrane forgrowing microorganisms in one or more discrete colonies, and theadsorption medium is in the form of a cohesive layer adjoining to andconformably disposed with the semipermeable medium and the support, theadsorption medium layer being removable from the semipermeable mediumand transferable for analysis to detect the presence of radioactivelylabeled metabolite and to identify specific colonies of microorganismsproducing the radioactively labeled metabolite.